Analogs of NT-3

ABSTRACT

The in vivo circulating life and/or absorption of Neurophic Factor-3 (NT-3) is increased by generating certain substitution analogs of the native protein sequence that result in a lower isoelectric point and charge in relation to NT-3 of native sequence.

This application is a division of application Ser. No. 09/255,953, filedFeb. 23, 1999, now U.S. Pat. No. 6,271,364, which is a continuation ofapplication Ser. No. 08/684,353, filed Jul. 19,1996, now abandoned,which are hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates generally to polypeptide analogs oftherapeutically active cationic proteins, including but not limited toanalogs of the neurotrophic factor known as NT-3. More specifically, theinvention relates to positively charged polypeptides in whichmodifications have been made in the native sequence, such that theanalogs possess lower isoelectric points and, concomitantly, longercirculation times and/or improved absorption in vivo followingparenteral administration. The invention also relates to materials andmethods for the recombinant production of such polypeptide analogs, toantibodies thereof, and to pharmaceutical compositions containing theanalogs which can be used for the treatment of various diseases anddisorders.

BACKGROUND OF THE INVENTION

Following the administration of a therapeutic protein by parenteralmeans, such as by subcutaneous, intravenous or intramuscular injection,the pharmacokinetic properties such as bioavailability, circulation timeand clearance rate can vary widely from protein to protein. Even thoughthere are active efforts in many laboratories to develop alternativeroutes of administration for protein products, little is known about thefactors that govern the pharmacokinetic behavior of protein therapeuticsfollowing such parenteral administration. It has been shown that anincrease in the molecular weight of a protein can result in apreferential uptake by the lymphatic system rather than the bloodcapillaries; Supersaxo et al., Pharmaceutical Res., Volume 7, page 167et seq. (1990). The molecular size of the therapeutic protein also playsa key role in insulin uptake, where dissociation of a zinc-inducedhexamer to monomeric form has been shown to be the rate-limiting step ininsulin absorbance; Kang et al., Diabetes Care, Volume 14, pages 942-948(1991). The clinical testing in diabetic patients of monomeric insulinanalogs, in which the hexamer association site has been eliminated, hasdemonstrated a more rapid uptake, leading to significant improvements inglucose control in diabetic patients; see Brange et al., Nature, Volume333, page 679 et seq. (1988).

SUMMARY OF THE INVENTION

To assess the impact of the isoelectric point (pI) on thepharmacokinetic behavior of proteins, certain analogs of NT-3, inparticular, have been produced which have a relatively lower pI, yetretain the structure and biological activity of the protein in its“native” state (i.e., the protein of naturally occurring amino acidsequence, as well as the met⁻¹ version thereof, both of which arereferred to herein as “wild type”). From these studies, it has now beendiscovered that protein analogs engineered to possess a lower pI and/orlower charge under physiological conditions than the wild type molecule,can also display longer in vivo circulation times (i.e., “half life”)and improved absorption following administration by injection. Althoughthe invention is illustrated in this description with particularreference to human NT-3, it has broader applicability to any cationicproteins, and particularly basic proteins which in their native sequencehave a pI greater than about 7.0.

It should be noted that the terms “protein” and “polypeptide” are usedinterchangeably throughout this description to mean one and the samething.

Briefly stated, the present invention is concerned with substitution,insertion and deletion analogs of cationic therapeutic proteins, and/orchemically modified versions of such therapeutic proteins, that arecharacterized by a lower pI while also exhibiting longer circulationtimes and/or higher absorption relative to the unmodified proteins(i.e., of native sequence). The analog proteins of this invention aretypically human therapeutic proteins which are usually, but notnecessarily, basic proteins. Preferably, these proteins also have alower charge under physiological conditions compared to the unmodifiedbasic protein.

The present invention also concerns materials and methods for therecombinant production of such analogs (as a preferred practicalmethod), as well as to antibodies raised against the protein analogs,and to pharmaceutical compositions containing the analogs asbiologically active agents for use in the treatment of diseases andphysical disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence for wild type human NT-3 (i.e., of“native” sequence) which has been produced recombinantly in E. colibacterial cells and expressed with a methionine residue present at theN-terminus, i.e., r-metHuNT-3 (SEQ ID NO: 1). In the Figure, the aminoacid numbering begins with the first residue after the initialmethionine (Met⁻¹), since the naturally occurring protein is normallyexpressed in mammalian cells without the methionine residue. (Note thatSequence Listing herein counts Met residue as +1).

FIGS. 2A and 2B show the nucleic acid sequence (SEQ ID NO: 2, and FIG.2A) and amino acid sequence (SEQ ID NO: 3, and FIG. 2B) of a polypeptideanalog of r-metHuNT-3 of the present invention, namely,NT-3₍₁₋₁₁₉₎R61A,K64D (with amino acid numbering in the Figure beginningwith first residue after initial methionine).

FIGS. 3A and 3B show the nucleic acid sequence (SEQ ID NO: 4, and FIG.3A) and amino acid sequence (SEQ ID NO: 5, and FIG. 3B) of anotherpolypeptide analog of r-metHuNT-3 according to the present invention,namely, NT-3₍₁₋₁₁₇₎R61A,K64D (amino acid numbering in Figure againbeginning with first residue after initial methionine).

FIG. 4 shows the ELISA assay calibration (standard) curves for wild typeNT-3 (i.e., having SEQ ID NO: 1) and for the NT-3 analogs of SEQ ID NOS:3 and 5. The optical density is plotted against sample concentration innanograms per milliliter (ng/ml). The cross-reactivity to NT-3 analogsin the ELISA assay is approximately ten percent. Serum samples frompharmacokinetic studies were analyzed by ELISA, and concentrations ofwild type NT-3 and NT-3 analogs were determined by calibration againstthe appropriate standard curve.

FIG. 5 depicts a size exclusion high performance liquid chromatogram(SEC-HPLC) in which wild type NT-3 is compared to the NT-3 analogs ofFIGS. 2 and 3. Units of absorbance (“AU”) are plotted on the verticalaxis, and the time of elution to the peak (in minutes) is shown on thehorizontal axis. As shown, the analogs co-elute with the wild typemolecule, demonstrating that the noncovalent dimer structure of wildtype NT-3 has not been disrupted in either of the analogs. No aggregatedprotein was detected in any of these preparations. Figure legends: ()wild type NT-3; () NT-3₍₁₋₁₁₇₎R61A,K64D; and () NT-3₍₁₋₁₁₉₎R61A,K64D.

FIG. 6 depicts a cation exchange high performance liquid chromatogram(CEX-HPLC) in which wild type NT-3 is compared to the NT-3 analogs ofFIGS. 2 and 3. “AU” (vertical axis) indicates absorbance units. Time ofelution to peak is shown on the horizontal axis. In this figure theanalogs elute sooner than wild type NT-3, which is consistent with thelower isoelectric points of the analogs. Figure legends: () wild typeNT-3; () NT-3₍₁₋₁₁₇₎R61A,K64D; and () NT-3₍₁₋₁₁₉₎R61A,K64D.

FIG. 7 depicts a silver stained SDS polyacrylamide gel electrophoresis(SDS-PAGE) chromatogram in which wild type NT-3 (see FIG. 1) is comparedto the NT-3 analogs of FIGS. 2 and 3. All three samples run as a singleband with approximately the same molecular weight of the monomeric form.None of the samples are seen to contain significant amounts of highermolecular weight oligomers or lower molecular weight fragments. Numberthe lanes from left to right starting with Lane 1. The lanes correspondto the following samples. Lane 1: NT-3₍₁₋₁₁₇₎R61A,K64D, 2.5 μg; Lane 2:NT-3₍₁₋₁₁₉₎R61A,K64D, 2.5 μg; Lane 3; wild type NT-3, 2.5 μg; Lane 4:wild type NT-3, 12.5 μg; Lane 5: molecular weight markers.

FIG. 8 shows the serum concentration (in nanograms per milliliter)versus time(in hours) profiles for the proteins of SEQ ID NOS: 1, 3 and5 after intravenous (IV) administration to test rats. The dose level was1 milligram per kilogram (mg/kg) of body weight. The concentrationprofiles are biphasic. The initial distribution phase was followed by aslower elimination phase. Each point on the graph represents an averageof three animals.

FIG. 9 shows the serum concentration (in nanograms per milliliter)versus time (in hours) curves in rats for the proteins of SEQ ID NOS: 1,3 and 5 following subcutaneous (SC) administration of 1 mg/kg, with eachpoint on the graph again representing an average of three animals. Theabsorption phase is characterized by an increase in serum concentrationto a peak. Wild type NT-3 (SEQ ID NO: 1) showed the most rapid declineafter attaining maximal concentration.

DETAILED DESCRIPTION OF THE INVENTION

The principles of this invention have broad applicability to anycationic proteins for which a reduction in the pI and, optionally,protein charge will result in an enhancement of therapeutically relevantbiological properties such as circulation time and/or absorptionfollowing parenteral administration. By way of illustration, suchproteins include but are not limited to basic proteins such asneurotrophic factor-3 (NT-3), brain derived neurotrophic factor (BDNF),megakaryocyte growth and development factor and various known isoformsthereof having essentially the same ability to increase blood plateletproduction in vivo and ex vivo (referred to herein collectively as“MGDF”), and keratinocyte growth factor (KGF). Detailed descriptions ofthese factors, their biological properties, and methods for theirpreparation and testing are given in the patent literature: NT-3 inpublished PCT application WO 91/03569; BDNF in U.S. Pat. Nos. 5,180,820,5,229,500, 5,438,121 and 5,453,361, and in published PCT application WO91/03568; MGDF in published PCT applications WO 95/26745, WO 95/21919,and WO 95/21920; and KGF in published PCT application WO 90/08771.

In essence, the objective of this invention is to make one or moremodifications to the primary structure of the wild type protein thatpreserve the protein structure and biological activity of the protein,but which also results in a lower isoelectric point and, preferably, alower charge at physiological pH. The particular way in which thesemodifications are made is not critical, and any procedure can be usedwhich effects the aforementioned changes to achieve the describedenhancements in properties. Merely by way of illustration, appropriatemodifications can be accomplished by use of site directed mutagenesisinvolving the addition of acidic residues to the sequence by insertionand/or replacement mutations, and/or removal of basic residues bydeletion and/or replacement mutations. Alternatively, chemical groups ormoieties can be added to selected sites (i.e., on amino acid residues)in the protein chain of the wild type molecule to accomplish the sameend purpose (i.e., reduction of pI and charge with preservation ofstructure and biological activity). A specific example is thesuccinylation of selected residues in the protein chain.

Nucleic acids which encode protein analogs in accordance with thisinvention (i.e., wherein one or more amino acids are designed to differfrom the wild type polypeptide) may be produced using site directedmutagenesis or PCR amplification in which the primer(s) have the desiredpoint mutations. For a detailed description of suitable mutagenesistechniques, see Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)and/or Ausubel et al., editors, Current Protocols in Molecular Biology,Green Publishers Inc. and Wiley and Sons, NY (1994). Chemical synthesisusing methods described by Engels et al. in Angew. Chem. Intl. Ed.,Volume 28, pages 716-734 (1989), may also be used to prepare suchnucleic acids.

The DNA molecules may be used to express the analog polypeptides of theinvention by recombinant methods familiar to those skilled in the art,including but not limited to methods described in the above mentionedpatents or patent applications for NT-3, BDNF, KGF and MGDF. By way ofillustration, a nucleic acid sequence encoding an analog polypeptide ofthis invention is inserted into an appropriate biologically functionalvector (e.g., circular plasmid or viral DNA) for expression in asuitable host cell. The vector includes regulatory sequences forexpression of the inserted nucleic acid sequence and is selected to befunctional in the particular host cell employed (i.e., the vector iscompatible with the host cell machinery, such that amplification and/orexpression of the gene can occur). The polypeptide may beamplified/expressed in prokaryotic, yeast, insect (baculovirus systems)and/or eukaryotic host cells. Selection of the host cell will depend atleast in part on whether the polypeptide expression product is to beglycosylated. If glycosylation is desired, then yeast, insect ormammalian host cells are preferred for use.

Typically, the vectors will contain a 5′ flanking sequence (alsoreferred to as a “promoter”) and other regulatory elements, as well asenhancer(s), an origin of replication element, a transcriptionaltermination element, a complete intron sequence containing a donor andacceptor splice site, a signal peptide sequence, a ribosome binding siteelement, a polyadenylation sequence, a polylinker region for insertingthe nucleic acid encoding the polypeptide to be expressed, and aselectable marker element.

The 5′ flanking sequence may be the innate 5′ flanking sequence from thewild type gene, or it may be homologous (i.e., from the same speciesand/or strain as the host cell), heterologous (i.e., from a speciesother than the host cell species or strain), hybrid (i.e., a combinationof 5′ flanking sequences from more than one source), or synthetic. Thesource of the 5′ flanking sequence may be any unicellular prokaryotic oreukaryotic organism, any vertebrate or invertebrate organism, or anyplant, provided that the 5′ flanking sequence is functional in, and canbe activated by, the host cell machinery.

The origin of replication element is typically a part of prokaryoticexpression vectors purchased commercially, and aids in the amplificationof the vector in a host cell. Amplification of the vector to a certaincopy number can, in some cases, be important for optimal expression ofthe polypeptide. If the vector of choice does not contain an origin ofreplication site, one may be chemically synthesized based on a knownsequence and then ligated into the vector.

The transcription termination element is typically located 3′ to the endof the polypeptide coding sequence and serves to terminate transcriptionof the polypeptide. Usually, the transcription termination element inprokaryotic cells is a G-C rich fragment followed by a poly-T sequence.While the element is easily cloned from a library or even purchasedcommercially as part of a vector, it can also be readily synthesizedusing methods for nucleic acid synthesis such as those referred toabove.

A selectable marker gene element encodes a protein necessary for thesurvival and growth of a host cell grown in a selective culture medium.Typical selection marker genes encode proteins that (a) conferresistance to antibiotics or other toxins, e.g., ampicillin,tetracycline, or kanamycin for prokaryotic host cells, (b) complementauxotrophic deficiencies of the cell; or (c) supply critical nutrientsnot available from complex media. Preferred selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene.

The ribosome binding element, commonly called the Shine-Dalgarnosequence (for prokaryotes) or the Kozak sequence (for eukaryotes), isnecessary for the initiation of translation for mRNA. The element istypically located 3′ to the promoter and 5′ to the coding sequence ofthe polypeptide to be synthesized. The Shine-Dalgarno sequence is variedbut is typically a polypurine (i.e., having a high A-G content). ManyShine-Dalgarno sequences have been identified, each of which can bereadily synthesized using methods set forth above and used in aprokaryotic vector.

In those cases where it is desirable for the polypeptide to be secretedfrom the host cell, a signal sequence may be used to direct thepolypeptide out of the host cell where it is synthesized. Typically, thesignal sequence is positioned in the coding region of nucleic acidsequence, or directly at the 5′ end of the coding region. Many signalsequences have been identified, and any of them that are functional inthe selected host cell may be used here. Consequently, the signalsequence may be homologous or heterologous to the polypeptide.Additionally, the signal sequence may be chemically synthesized usingmethods such as those referred to set above.

Host cells may be prokaryotic host cells (such as E. coli) or eukaryotichost cells (such as yeast, insect or vertebrate cells). The host cell,when cultured under suitable nutrient conditions, can synthesize thepolypeptide, which can subsequently be collected by isolation from theculture medium (if the host cell secretes it into the medium) ordirectly from the host cell producing it (if not secreted). Aftercollection, the polypeptide can be purified using methods such asmolecular sieve chromatography, affinity chromatography, and the like.In general, if the polypeptide is expressed in E. coli it will contain amethionine residue at the N-terminus in its recovered form (i.e. met⁻¹),unless expressed in a strain of E. coli in which the methionine isenzymatically cleaved off by the host.

Suitable cells or cell lines may also be mammalian cells, such asChinese hamster ovary cells (CHO) or 3T3 cells. The selection ofsuitable mammalian host cells and methods for transformation, culture,amplification, screening and product production and purification areknown in the art. Other suitable mammalian cell lines are the monkeyCOS-1 and COS-7 cell lines, and the CV-1 cell line. Further exemplarymammalian host cells include primate cell lines and rodent cell lines,including transformed cell lines. Normal diploid cells, cell strainsderived from in vitro culture of primary tissue, as well as primaryexplants, are also suitable. Candidate cells may be genotypicallydeficient in the selection gene, or they may contain a dominantly actingselection gene. Still other suitable mammalian cell lines include butare not limited to, HeLa, mouse L-929 cells, 3T3 lines derived fromSwiss, Balb-c or NIH mice, BHK or HaK hamster cell lines.

Insertion (also referred to as “transformation” or “transfection”) ofthe vector into the selected host cell may be accomplished using calciumchloride, electroporation, microinjection, lipofection or theDEAE-dextran method. The method selected will in part be a function ofthe type of host cell to be used. These methods and other suitablemethods are well known to the skilled artisan, and are set forth, forexample, in Sambrook et al., above.

The host cells containing the vector may be cultured using standardmedia well known to the skilled artisan. The media will usually containall of the nutrients necessary for the growth and survival of the cells.Suitable media for culturing E. coli cells are, for example, Luria Broth(LB) and/or Terrific Broth (TB). Suitable media for culturing eukaryoticcells are RPMI 1640, MEM, DMEM, all of which may be supplemented withserum and/or growth factors as required by the particular cell linebeing cultured. A suitable medium for insect cultures is Grace's mediumsupplemented with yeastolate, lactalbumin hydrolysate and/or fetal calfserum as necessary.

Typically, an antibiotic or other compound useful for selective growthof the transformed cells is added as a supplement to the media. Thecompound to be used will be dictated by the selectable marker elementpresent on the plasmid with which the host cell was transformed. Forexample, where the selectable marker element is kanamycin resistance,the compound added to the culture medium will be kanamycin.

The amount of polypeptide produced in the host cell can be evaluatedusing standard methods known in the art. Such methods include, withoutlimitation, Western blot analysis, SDS-polyacrylamide gelelectrophoresis, non-denaturing gel electrophoresis, HPLC separation,immunoprecipitation, and/or activity assays such as DNA binding gelshift assays.

If the polypeptide has been designed to be secreted from the host cells,the majority of polypeptide will likely be found in the cell culturemedium. If, however, the polypeptide is not secreted, it will be presentin the cytoplasm (for eukaryotic, Gram-positive bacteria, and insecthost cells) or in the periplasm (for Gram-negative bacteria host cells).

For intracellular polypeptide, the host cells are typically firstdisrupted mechanically or osmotically to release the cytoplasmiccontents into a buffered solution. The polypeptide is then isolated fromthis solution. Purification of the polypeptide from solution canthereafter be accomplished using a variety of techniques. If thepolypeptide has been synthesized such that it contains a tag such asHexahistidine or other small peptide at either its carboxyl or aminoterminus, it may be purified in a one-step process by passing thesolution through an affinity column where the column matrix has a highaffinity for the tag or for the polypeptide directly (i.e., a monoclonalantibody). For example, polyhistidine binds with great affinity andspecificity to nickel, thus an affinity column of nickel (such as theQiagen nickel columns) can be used for purification. (See, for example,Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley& Sons, New York, 1994).

Where, on the other hand, the polypeptide has no tag and no antibodiesare available, other well known procedures for purification can be used.Such procedures include, without limitation, ion exchangechromatography, molecular sieve chromatography, HPLC, native gelelectrophoresis in combination with gel elution, and preparativeisoelectric focusing (“Isoprime” machine/technique, Hoefer Scientific).In some cases, two or more of these techniques may be combined toachieve increased purity.

If it is anticipated that the polypeptide will be found primarily in theperiplasmic space of the bacteria or the cytoplasm of eukaryotic cells,the contents of the periplasm or cytoplasm, including inclusion bodies(e.g., Gram-negative bacteria) if the processed polypeptide has formedsuch complexes, can be extracted from the host cell using any standardtechnique known to the skilled artisan. For example, the host cells canbe lysed to release the contents of the periplasm by the use of a Frenchpress, homogenization, and/or sonication. The homogenate can then becentrifuged.

In addition to preparing the polypeptide analogs of this invention byrecombinant DNA techniques, the polypeptides may be prepared by chemicalsynthesis methods (such as solid phase peptide synthesis) usingtechniques known in the art, including those set forth by Merrifield etal. in J. Am. Chem. Soc., Volume 85, page 2149 (1964), by Houghten etal. in Proc. Natl. Acad. Sci. USA, Volume 82, page 5132 (1985), and byStewart and Young in Solid Phase Peptide Synthesis, Pierce Chem. Co,Rockford, Ill. (1984). Chemically synthesized polypeptides may beoxidized using methods set forth in these references to form disulfidebridges.

The pI and charge of the protein analogs resulting from any of theaforementioned methods can be measured using standard techniques, suchas those described further below in conjunction with the specificembodiments.

Chemically modified polypeptide compositions (i.e., “derivatives”) wherethe polypeptide is linked to a polymer in order to modify properties areincluded within the scope of the present invention. The polymer istypically water soluble so that the protein to which it is attached doesnot precipitate in an aqueous environment, such as a physiologicalenvironment. The polymer may have a single reactive group, such as anactive ester for acylation or an aldehyde for alkylation, so that thedegree of polymerization may be controlled. A preferred reactivealdehyde is polyethylene glycol propionaldehyde, which is water stable,or mono C1-C10 alkoxy or aryloxy derivatives thereof (see U.S. Pat. No.5,252,714). The polymer may be branched or unbranched. Preferably, fortherapeutic use of the end-product preparation, the polymer will bepharmaceutically acceptable. The water soluble polymer, or mixturethereof if desired, may be selected from the group consisting of, forexample, polyethylene glycol (PEG), monomethoxy-polyethylene glycol,dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, apolypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols(e.g., glycerol) and polyvinyl alcohol.

In general, the polypeptide analogs of this invention will be useful forthe same purposes for which the wild type proteins from which they arederived are known to be useful. For instance, NT-3 is currently underclinical study for the treatment of peripheral (including diabetic)neuropathies, while BDNF is under clinical study for the treatment ofamyotrophic lateral sclerosis (ALS). KGF is known to be active as atissue growth and repair factor, and is currently in human clinicaldevelopment for the treatment of chemotherapy- or radiation-inducedmucositis. MGDF (in the form of a pegylated derivative) is in clinicaldevelopment for the stimulation of platelet production as an adjunct tochemotherapy-induced thrombocytopenia. However, it is expected that theanalogs of this invention will offer advantages over the unmodifiedforms from the standpoint of enhanced therapeutic half life andabsorbability.

For therapeutic purposes, the analog polypeptides of this invention willtypically be formulated into suitable pharmaceutical compositionsadapted for therapeutic delivery, which constitutes an additional aspectof this invention. Such pharmaceutical compositions will typicallycomprise a therapeutically active amount of an analog polypeptide, aloneor together with one or more excipients, carriers, or other standardingredients for a pharmaceutical composition. The carrier material maybe water for injection, preferably supplemented with other materialscommon in solutions for administration to mammals. Typically, the analogpolypeptide will be administered in the form of a composition comprisinga purified form of the polypeptide (which may be chemically modified) inconjunction with one or more physiologically acceptable carriers,excipients, or diluents. Neutral buffered saline or saline mixed withserum albumin are exemplary appropriate carriers. Other standardcarriers, diluents, and excipients may be included as desired.

The pharmaceutical compositions of this invention may be prepared forstorage by mixing the selected composition having the desired degree ofpurity with optional physiologically acceptable carriers, excipients, orstabilizers (Remington's Pharmaceutical Sciences, 18th edition, A. R.Gennaro, ed., Mack Publishing Company, 1990) in the form of alyophilized cake or an aqueous solution. Acceptable carriers, excipientsor stabilizers are nontoxic to recipients and are preferably inert atthe dosages and concentrations employed, and include buffers such asphosphate, citrate, acetate, succinate or other organic acid salts;antioxidants such as ascorbic acid; low molecular weight polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as Tween, Pluronics or polyethylene glycol (PEG).

Any composition of this invention which is intended to be used for invivo administration must be sterile. Sterilization is readilyaccomplished by filtration through sterile filtration membranes. Wherethe composition is lyophilized, sterilization using these methods may beconducted either prior to, or following, lyophilization andreconstitution. The composition for parenteral administration ordinarilywill be stored in lyophilized form or in solution.

The amount of polypeptide that will be effective in the treatment of aparticular disorder or condition will depend on the nature of thepolypeptide and disorder or condition, as well as the age and generalhealth of the recipient, and can be determined by standard clinicalprocedures. Where possible, it will be desirable to determine thedose-response curve of the pharmaceutical composition first in vitro, asin bioassay systems, and then in useful animal model systems in vivoprior to testing in humans. In general, suitable in vivo amounts can bedeveloped based on a knowledge of the therapeutically effective dosesknown for the wild type protein on which the analogs are based. Theskilled practitioner, considering the therapeutic context, type ofdisorder under treatment, etc., will be able to ascertain proper dosingwithout undue effort.

Methods of introduction for administration purposes include intradermal,intramuscular, intraperitoneal, intravenous, and subcutaneous. Inaddition, the invention also encompasses pharmaceutical compositionscomprising the polypeptide analogs administered via liposomes,microparticles or microcapsules, which may be particularly useful toachieve sustained release.

Special delivery devices may needed in the case of some of thepolypeptide analogs, such as those of NT-3, BDNF and other neurotrophicfactors intended for the treatment of neurological conditions associatedwith the brain and other areas of the central nervous system. Suchdevices may include implants and osmotic pumps for intrathecal andintracranial delivery, for instance.

The analogs of this invention can also be used in accordance withstandard procedures to generate antibodies that are useful for medicallyrelated purposes, such as for the monitoring of blood levels of thecorresponding analog in a subject undergoing therapeutic treatment.Various procedures known in the art can be employed for the productionof polyclonal antibodies that recognize epitopes of the polypeptides.For the production of antibody, various host animals can be immunized byinjection with an analog polypeptide, or fragment or derivative thereof,including but not limited to rabbits, mice, rats, etc. Various adjuvantsmay be used to increase the immunological response, depending on thehost species, including but not limited to Freund's, mineral gels suchas aluminum hydroxide (alum), surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum.

For the preparation of monoclonal antibodies directed toward the analogpolypeptides, any technique which provides for the production ofantibody molecules by continuous cell lines in culture may be used. Forexample, the hybridoma technique originally developed by Kohler andMilstein and described in Nature, Volume 256, pages 495-497 (1975), aswell as the trioma technique, the human B-cell hybridoma techniquedescribed by Kozbor et al in Immunology Today, Volume 4, page 72 (1983),and the EBV-hybridoma technique to produce monoclonal antibodiesdescribed by Cole et al in “Monoclonal Antibodies and Cancer Therapy”,Alan R. Liss, Inc., pages 77-96 (1985), are all useful for preparationof monoclonal antibodies.

In addition, a molecular clone of an antibody to an epitope or epitopesof the polypeptide can be prepared with known techniques. In particular,recombinant DNA methodology may be used to construct nucleic acidsequences which encode a monoclonal antibody molecule or antigen-bindingregion thereof; see, for example, Maniatis et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1982).

The antibodies are useful for both in vivo and in vitro diagnosticpurposes, particularly in labeled form to detect the presence of thepolypeptides in a fluid or tissue sample.

DESCRIPTION OF SPECIFIC EMBODIMENTS

The invention is described in further detail with reference to thefollowing materials, methods, procedures and test results. Amino acidresidues of proteins are identified the in conventional manner using theestablished three letter designations (for example, “met” formethionine, “val” for valine, etc.) or in some cases the establishedsingle letter designations (for example, “M” for methionine, “R” forarginine, etc.) throughout the text.

Materials and Methods

Preparation of NT-3 Analogs

Amino acid residues for substitution in the native sequence of humanNT-3 were selected with the aim of preserving the core structure andbiological/therapeutic activity (see Holland et al., J. Mol. Biol.,Volume 239, pages 385-400, 1994; Ibanez et al., in Cell, Volume 69,pages 329-341, 1992 and also in EMBO Journal, Volume 12, pages2281-2293, 1993). The actual substitutions that were made in the nativesequence of human NT-3 are reflected in the sequences shown in FIGS. 2and 3, respectively. In one analog, shown in FIG. 2, the following twosubstitutions were made: arginine at position 61 (arg₆₁) was replaced byalanine (ala), and lysine at position 64 (lys₆₄) was replaced byaspartic acid (asp). This analog was designated “NT-3₍₍₁₋₁₁₉₎R61A,K64D”. A second analog, shown in FIG. 3, had these same two aminoacid substitutions and, in addition, was truncated at residue 117 (thusdeleting arg₁₁₈ and thr₁₁₉). This analog was designated“NT-3₍₁₋₁₁₇₎R61A,K64D”. To create these analogs, the mutations wereintroduced in the sequence of human NT-3 by standard Polymerase ChainReaction (PCR) technology. For NT-3₍ 1-119)R61A,K64D, chemicallysynthesized oligonucleotides were used in pairs to create fragments ofthe NT-3 gene comprising the front portion up to the site of themutations at the codons corresponding to positions 61 and 64 and theback portion of the gene from the mutant codons to the end. A second PCRwas carried out combining the front and back portions to create the fulllength nucleic acid molecule encoding the two mutations at positions 61and 64, respectively. For NT-3₍ 1-117)R61A,K64D, the foregoing procedurewas repeated, except the back portion omitted the codons for arginineand threonine at positions 118 and 119.

Expression in E. coli

To express the analogs in E. coli, a DNA sequence encoding for amethionine residue was included at the 5′ end and a stop codon wasplaced at the 3′ end in each case. In addition, cutting sites for therestriction enzymes XbaI and HindIII were placed at the extreme 5′ and3′ ends of the gene, respectively, and a synthetic ribosome binding sitewas placed an appropriate distance upstream of the initiatingmethionine. The resulting synthetic gene fragments, flanked by XbaI andHindIII restriction sites at the 5′ and 3′ ends, respectively, containeda ribosome binding site, the ATG start codon (encoding methionine), thesequence encoding the analog, and a stop codon. The fragments weredigested with restriction endonucleases NdeI and BamHI, and then ligatedinto the vector pAMG12.

The expression plasmid pAMG12 can be derived from the plasmid pCFM1656(ATCC Accession No. 69576, deposited Feb. 24, 1994) by making a seriesof site directed base changes by PCR overlapping oligo mutagenesis andDNA sequence substitutions. Starting with the BglII site (plasmid basepair no. 180) immediately 5′ to the plasmid replication promoterP_(copB) and proceeding toward the plasmid replication genes, the basepair (bp) changes are as follows:

bp in pCFM1656 pAMG12 bp no. pAMG12 bp changed to in #204 T/A C/G #428A/T G/C #509 G/C A/T #617 — insert two G/C bp #679 G/C T/A #980 T/A C/G#994 G/C A/T #1004 A/T C/G #1007 C/G T/A #1028 A/T T/A #1047 C/G T/A#1178 G/C T/A #1466 G/C T/A #2028 G/C bp deletion #2187 C/G T/A #2480A/T T/A #2499-2502 AGTG GTCA TCAC CAGT #2642 TCCGAGC bp deletion AGGCTCG#3435 G/C A/T #3446 G/C A/T #3643 A/T T/A

In addition, the DNA sequence between the unique AatII (position #4364in pCFM1656) and SacII (position #4585 in pCFM1656) restriction sites issubstituted with the following DNA sequence (SEQ ID NO: 8 and SEQ ID NO:9):

[AatII sticky end] 5′     CGTAACGTATGCATGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAA- 3′GCACGCATTGCATACGTACCAGAGGGGTACGCTCTCATCCCTTGACGGTCCGTAGTT--TAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTG--ATTTTGCTTTCCGAGTCAGCTTTCTGACCCGGAAAGCAAAATAGACAACAAACAGCCAC--ACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGG--TGCGAGAGGACTCATCCTGTTTAGGCGGCCCTCGCCTAAACTTGCAACGCTTCGTTGCC--CCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAG--GGCCTCCCACCGCCCGTCCTGCGGGCGGTATTTGACGGTCCGTAGTTTAATTCGTCTTC--CCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTGTTTATTTTTCTAAAT--CGGTAGGACTGCCTACCGGAAAAACGCAAAGATGTTTGAGAAAACAAATAAAAAGATTTA-             AatII-ACATTCAAATATGGACGTCTCATAATTTTTAAAAAATTCATTTGACAAATGCTAAAATTC--TGTAAGTTTATACCTGCAGAGTATTAAAAATTTTTTAAGTAAACTGTTTACGATTTTAAG--TTGATTAATATTCTCAATTGTGAGCGCTCACAATTTATCGATTTGATTCTAGATTTGAGT--AACTAATTATAAGAGTTAACACTCGCGAGTGTTAAATAGCTAAACTAAGATCTAAACTCA--TTTAACTTTTAGAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGAGCTCACTAGT--AAATTGAAAATCTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGCTCGAGTGATCA-                                   SacII-GTCGACCTGCAGGGTACCATGGAAGCTTACTCGAGGATCCGCGGAAAGAAGAAGAAGAAG--CAGCTGGACGTCCCATGGTACCTTCGAATGAGCTCCTAGGCGCCTTTCTTCTTCTTCTTC--AAGAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAA--TTCTTTCGGGCTTTCCTTCGACTCAACCGACGACGGTGGCGACTCGTTATTGATCGTATT--CCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACCGCTCTT--GGGGAACCCCGGAGATTTGCCCAGAACTCCCCAAAAAACGACTTTCCTCCTTGGCGAGAA--CACGCTCTTCACGC 3′ -GTGCGAGAAGTG   5′

[SacII Sticky End]

The ligation product was transformed into competent host cells of E.coli strain FM15. Resulting colonies were screened for the production ofrecombinant protein and those colonies producing the correct-sizedprotein were verified by DNA sequencing. The correct strain wasinoculated for fermentation by transferring a small amount to LuriaBroth (10 g/l of Trypticase-Peptone, 10 g/l of Yeast Extract, and 5 g/lof sodium chloride) and incubating at 30° C. for sixteen hours withstirring at 250 rpm. The culture was transferred to sterile medium thathad been sterilized in place in a fermentor, then the mass of the cellswas increased using continuous feeds of glucose and organic nitrogen,before being induced with lactose. After induction, the fermentation washalted, the cells were harvested by centrifugation, the supernatant wasremoved, and the remaining cell paste was frozen.

Protein Purification

Cells from the paste were broken by high pressure homogenization andinclusion bodies were recovered by centrifugation. The inclusion bodieswere solubilized in guanidine-HCl, then diluted into urea. Afterstanding for several days, the solution was adjusted to pH 3, dilutedwith water, centrifuged, and eluted in series through cation exchangeand hydrophobic interaction chromatography columns. The peak fractionswere pooled and sterile filtered.

Isoelectric Point and Protein Charge

The isoelectric points of wild type human NT-3 (r-metHuNT-3, SEQ IDNO:1), and of the analogs thereof (i.e., SEQ ID NOS: 3 and 5) werecalculated using the “GCG” protein/DNA Sequence Analysis SoftwarePackage available from Genetics Computer Group, Inc., Madison, Wis. Thecharge of the molecule at physiological pH (assumed to be pH 7.4) wasestimated using the same software. The pI of the first analog,NT-3₍₁₋₁₁₉₎R61A,K64D (SEQ ID NO: 3), was calculated to be about 0.9 pHunits below that of wild type NT-3 (8.5, compared to 9.4). The charge atphysiologic pH (7.4) for this analog represented a reduction of about2.5 pH units from that of wild type NT-3 (i.e., from approximately +7 to+4.5). The pI for the other analog, NT-3₍ ₁₋₁₁₇₎R61A,K64D (SEQ ID NO: 5)was calculated to be approximately 8.2, which was about 1.2 pH unitslower than the pI of wild type NT-3 (9.4). Moreover, the charge atphysiologic pH for this analog was decreased by approximately 3.5 pHunits, to about +3.5, relative to wild type NT-3 (+7).

ELISA Assay

The ELISA assay was conducted on 96-well plates coated with a monoclonalantibody raised against human NT-3. A rabbit polyclonal antibodyconjugated to horse radish peroxidase was used as the secondaryantibody. Serum samples, calibration standards, and quality controlsamples were diluted with phosphate buffer to a 50% serum matrix beforeassay. The sample size was 100 microliters (μl) per well. Each samplewas assayed in duplicate. The limits of quantification were 0.65, 4.00,and 4.05 ng/ml of serum for wild type NT-3, NT-3₍₁₋₁₁₉₎R61A,K64D andNT-3₍ ₁₋₁₁₇₎R61A,K64D, respectively.

Size Exclusion Chromatography (SEC-HPLC)

Size exclusion chromatography on each sample was performed using aWaters 600 system in conjunction with a G2000SWXL column (TosoHaas).Samples were eluted at a flow rate of 0.7 milliliters per minute(ml/min) in a buffer consisting of 100 mM sodium phosphate, 0.5 M NaCl,pH 6.09. Peaks were detected at a wavelength of 230 nanometers (nm).

Cation Exchange Chromatography (CEX-HPLC)

CEX-HPLC was performed using a Waters 625 system with a Resource Scolumn (Pharmacia, Uppsala, Sweden). Samples were eluted at a flow rateof one milliliter per minute using a sodium chloride gradient from 0-1 Min 20 mM Tris HCl, pH 8.5. Peaks were detected using a wavelength of 220nm.

Silver Stained SDS-PAGE

Proteins were diluted with 2% SDS, mixed with sample buffer, and heatedfor five minutes in boiling water. The separation was conductedaccording to manufacturer's instructions using precast TRIS-Tricinegradient gels, 10-20%, from ISS (Integrated Separations Systems, Natick,Mass.). Silver staining was done according to the procedure of Blum etal. in Electrophoresis, Volume 8, pages 93-99 (1987).

Mitogenic Bioassay With 3T3trkC Cells

The biological activity of r-metHuNT-3 as a reference standard isdetermined by means of a cell mitogenic bioassay utilizing 3T3trkCcells. These cells are created by transfecting 3T3 cells (ATCC), whichnormally do not express trkC receptor on their surface, with plasmidpcDNA1/neo (Invitrogen, San Diego, Calif.) modified to contain the DNAsequence for human trkC receptor protein. See Shelton et al., Journal ofNeuroscience, Volume 15, pages 477-491 (1995) for the sequence of thetrkC gene, and Valenzuela et al., Neuron, Volume 10, pages 963-974(1993) for an illustrative transfection procedure. The transfected cellsare maintained at 37±2° C., in a high humidity incubator under anatmosphere containing 5.5±1.0% CO₂ and in Dulbecco's Minimum EssentialMedium with fetal bovine serum and G-418 Sulfate. The cells aredistributed into 96-well plates for each assay. After approximatelytwenty four hours of incubation time under the same conditions, themaintenance medium is replaced with RPMI 1640 and test samples areadded. Varying concentrations of a standard and a test sample ofr-metHuNT-3 are prepared in RPMI 1640 and added to the appropriatewells. The plates are returned to the incubator for approximately twentyfour hours, then the cells are stained for viability with3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazoliumsalt (MTS), a tetrazolium compound. The plates are returned to theincubator for approximately five hours, after which the optical densityof each well is read at 490 nm. A dose-response standard curve isconstructed and a linear regression analysis is performed on the linearportion of the standard curve. The concentration of test sampledilutions falling within the linear extracted range of the standardcurve are then determined.

In Vivo Biological Testing

The effect of amino acid changes on the biological properties of amolecule was evaluated in vivo in male Sprague Dawley rats, using a“cross-over” test design. In particular, nine animals were divided intothree groups of three rats each, and the rats were administered wildtype NT-3, NT-3₍₁₋₁₁₉₎ R61A,K64D, or NT-3₍₁₋₁₁₇₎R61A,K64D at a dose of 1milligram per kilogram of body weight (mg/kg) each. The test materialwas administered either (1) intravenously (IV) as a first dose, thensubcutaneously (SC) at twenty four hours after the first dose, or (2) inthe reverse manner. Serial blood samples were collected before dosingand at 1, 5, 15, and 30 minutes, and 1, 2, 4, and 8 hours after an IVdose. Following subcutaneous dosing, samples were collected at 10 and 30minutes, and at 1, 2, 4, and 8 hours. Blood serum concentrations of NT-3were determined using an ELISA assay (see above). The antibodies used inthe assays were specific for quantitation of wild type NT-3. Though notfully optimized, the antibodies displayed sufficient cross-reactivitywith NT-3₍₁₋₁₁₉₎R61A,K64D and NT-3₍₁₋₁₁₇₎ R61A,K64D to allow forquantitation of these analogs also. Standard curves were prepared foreach protein (see FIG. 4).

Test Results

Characterization of NT-3 Analogs

Both of the analogs, NT-3₍₁₋₁₁₉₎R61A,K64D and NT-3₍ 1-117) R61A,K64D,were seen to retain the activity of wild-type NT-3 (r-metHuNT-3) in thePC-12 in vitro bioassay (Table 1). In fact, the bioactivity of theanalogs as evaluated in this assay appeared to be somewhat greater thanthat of wild-type NT-3, perhaps indicating an increased affinity for thetrkC (NT-3) receptor.

TABLE 1 In Vitro Bioactivity of NT-3 Proteins Percentage ofConcentration Measured expected submitted concentration activity NT-3Analog (mg/ml) (mg/ml) (%) Wild-type 0.32 0.23 72 NT-3 NT-3₍₁₋₁₁₇₎ 0.361.18 328 R61A,K64D NT-3₍₁₋₁₁₉₎ 0.25 1.31 524 R61A,K64D

Both analogs eluted as noncovalent dimers on size exclusion-HPLC, thesame as for wild type NT-3 (see FIG. 5). As expected, there was nosignificant difference in the molecular weights of the three proteins.No significant protein aggregation was detected for either analog. Theresults of cation exchange-HPLC (see FIG. 6) were consistent with areduction in pI for both analogs. The change in protein charge probablyaccounts for slight shifts in the SDS-PAGE (FIG. 7), compared to wildtype NT-3. Both of the analogs retain the biological activity andnoncovalent dimer structure of wild type NT-3.

Pharmacokinetic Behavior

Serum concentration curves following intravenous administration wereseen to be biphasic (see FIG. 8). There was a significant difference inthe initial distribution phase among the various NT-3 types. The halflifes (αT_(1/2)) were 3.3, 5.4 and 7.6 minutes for wild type NT-3,NT-3₍₁₋₁₁₉₎R61A,K64D, and NT-3₍₁₋₁₁₇₎ R61A,K64D, respectively (see Table2, below). The observed decrease in clearance following intravenousadministration could be due solely to the slower distribution of theNT-3 analogs. The most pronounced decrease in concentration during thisphase was observed with wild type NT-3. The terminal phase half lives(βT_(1/2)) were similar for the three types and ranged from 0.9 to 1.0hours, suggesting that the elimination mechanism for these types couldbe the same. The areas under the concentration-time curves (t-AUCinf)for NT-3₍₁₋₁₁₇₎R61A,K64D and NT-3₍₁₋₁₁₉₎ R61A,K64D were approximately1.2 to 2-fold that of wild type NT-3.

TABLE 2 Pharmacokinetic Parameters Obtained in Rats Given a Single IVDose of 1 mg/kg of NT-3 Proteins NT-3 t-AUC(inf)¹ CL² αT_(1/2) ³βT_(1/2) ⁴ Type (ng-hr/ml) (ml/kg-hr) (min) (hr) Wild  724.1 ± 1411.7 ±3.3 ± 1.0 ± type 151.0 294.4 0.3 0.6 NT-3₍₁₋₁₁₉₎  880.7 ± 1276.2 ± 5.4 ±0.9 ± R61A,K64D 320.3 581.8 1.9 0.6 NT-3₍₁₋₁₁₇₎ 1420.7 ±  706.3 ± 7.6 ±1.0 ± R61A,K64D 117.9 58.7 0.0 0.0 ¹Area under the serum concentrationtime curve from time zero to infinity. Area calculation is bytrapezoidal method. ²Clearance rate is calculated by: Dose ÷ AUC.³Distribution phase half life (αT_(1/2)). ⁴Terminal phase half life(βT_(1/2)).

Following subcutaneous injection, the serum concentration increasesrapidly for wild type NT-3 (see FIG. 9). The time to maximumconcentration (T_(MAX)) was approximately 0.17 hour (see Table 3,below). A slower absorption profile was observed for the modified types.Specifically, T_(MAX)'s for NT-3₍₁₋₁₁₉₎ R61A,K64D and NT-3₍₁₋₁₁₇₎R61A,K64D were 0.67 and 1.33 hours, respectively. The maximumconcentrations for NT-3₍₁₋₁₁₉₎R61A,K64D and NT-3₍₁₋₁₁₇₎ R61A,K64D were 6to 10 times higher than for wild type NT-3, suggesting that the analogsmanifested a greater degree of absorption from the site of injectionfollowing administration. Furthermore, the degree or extent ofabsorption (bioavailability) is more typically determined from the ratioof the areas under the serum curves for subcutaneous and intravenousroutes of administration, Table 3. The bioavailability for wild typeNT-3, NT-3₍₁₋₁₁₉₎ R61A,K64D, and NT-3₍₁₋₁₁₇₎ R61A,K64D was 2.2, 28.5 and43.22%, respectively. The terminal half lifes for the analogs appear tobe longer than that of the wild type.

TABLE 3 Pharmacokinetic Parameters Obtained in Rats Given a Single SCDose of 1 mg/kg of NT-3 Proteins NT-3 t-AUC(inf)¹ F² CMAX³ TMAX⁴βT_(1/2) ⁵ Type (ng-hr/ml) (%) (ng/ml) (hr) (hr) Wild  15.2 ±  2.2 ± 17.8 ± 0.2 ± 0.4 ± type 9.5 1.3 11.6 0.0 0.0 NT-3₍₁₋₁₁₉₎ 241.3 ± 28.5 ±106.5 ± 0.7 ± 1.4 ± R61A,K64D 74.8 6.3 47.1 0.3 0.6 NT-3₍₁₋₁₁₇₎ 632.9 ±43.2 ± 180.1 ± 1.3 ± 1.4 ± R61A,K64D 38.0 2.0 51.2 0.6 0.8 ¹Area underthe serum concentration time curve from time zero to infinity. Areacalculation is by trapezoidal method. ²Bioavailability (F) is calculatedby: (t-AUC_(SC) ÷ t-AUC_(IV)) × 100%, where both areas were obtainedfrom the same animal. ³CMAX is the maximal concentration. ⁴TMAX is thetime to maximal concentration. ⁵Terminal phase half life (βT_(1/2)).

The results from these studies show that by decreasing the pI of NT-3,one can lower the clearance rate following intravenous administration,at least initially, and also enhance the extent of absorption followingsubcutaneous administration. These results also demonstrate that thecharge of the protein plays a significant role in determiningpharmacokinetic behavior. In the case of a basic protein such as NT-3,as well as other cationic proteins, decreasing the isoelectric point (orthe charge at physiological pH) can lead to significant improvement inthe absorption and bioavailability of the molecule followingsubcutaneous administration. From this knowledge, and the descriptionprovided herein, it is possible to design new molecules of improvedtherapeutic value.

It should be noted that the analogs illustrated in the foregoingdescription are intended to be exemplary only, and that additionalanalogs of NT-3, as well as of other proteins, can be created in lightof the present description to achieve lower isolectric points withlonger circulation times and/or higher absorption. In one variation, forinstance, the particular analog proteins of SEQ ID NOS: 3 and 5 can beproduced by expressed in a mammalian cell or a secreting bacterialstrain such that a “met-less” product is obtained (i.e., the methionineresidue at the N-terminus is processed away, to result in thepolypeptides of SEQ ID NOS: 6 and 7, respectively).

The invention is defined in the appended claims.

9 1 120 PRT Homo sapiens 1 Met Tyr Ala Glu His Lys Ser His Arg Gly GluTyr Ser Val Cys Asp 1 5 10 15 Ser Glu Ser Leu Trp Val Thr Asp Lys SerSer Ala Ile Asp Ile Arg 20 25 30 Gly His Gln Val Thr Val Leu Gly Glu IleLys Thr Gly Asn Ser Pro 35 40 45 Val Lys Gln Tyr Phe Tyr Glu Thr Arg CysLys Glu Ala Arg Pro Val 50 55 60 Lys Asn Gly Cys Arg Gly Ile Asp Asp LysHis Trp Asn Ser Gln Cys 65 70 75 80 Lys Thr Ser Gln Thr Tyr Val Arg AlaLeu Thr Ser Glu Asn Asn Lys 85 90 95 Leu Val Gly Trp Arg Trp Ile Arg IleAsp Thr Ser Cys Val Cys Ala 100 105 110 Leu Ser Arg Lys Ile Gly Arg Thr115 120 2 360 DNA Artificial Sequence Description of Artificial SequenceAnalog of human NT-3 2 atgtacgctg aacacaaatc tcaccgtggt gaatactctgtttgcgactc tgaatctctg 60 tgggttaccg acaaatcttc tgctatcgac atccgtggtcaccaggttac cgttctgggt 120 gaaatcaaaa ccggtaactc tccggttaaa cagtacttctacgaaacccg ttgcaaagaa 180 gctgcaccgg ttgacaacgg ttgccgtggt atcgacgacaaacactggaa ctctcagtgc 240 aaaacctctc agacctacgt tcgtgctctg acctctgaaaacaacaagct tgttggttgg 300 cgttggattc gtatcgacac ctcttgcgtt tgcgctctgtctcgtaaaat cggtcgtacc 360 3 120 PRT Artificial Sequence Description ofArtificial Sequence Analog of human NT-3. 3 Met Tyr Ala Glu His Lys SerHis Arg Gly Glu Tyr Ser Val Cys Asp 1 5 10 15 Ser Glu Ser Leu Trp ValThr Asp Lys Ser Ser Ala Ile Asp Ile Arg 20 25 30 Gly His Gln Val Thr ValLeu Gly Glu Ile Lys Thr Gly Asn Ser Pro 35 40 45 Val Lys Gln Tyr Phe TyrGlu Thr Arg Cys Lys Glu Ala Ala Pro Val 50 55 60 Asp Asn Gly Cys Arg GlyIle Asp Asp Lys His Trp Asn Ser Gln Cys 65 70 75 80 Lys Thr Ser Gln ThrTyr Val Arg Ala Leu Thr Ser Glu Asn Asn Lys 85 90 95 Leu Val Gly Trp ArgTrp Ile Arg Ile Asp Thr Ser Cys Val Cys Ala 100 105 110 Leu Ser Arg LysIle Gly Arg Thr 115 120 4 354 DNA Artificial Sequence Description ofArtificial Sequence Analog of human NT-3. 4 atgtacgctg aacacaaatctcaccgtggt gaatactctg tttgcgactc tgaatctctg 60 tgggttaccg acaaatcttctgctatcgac atccgtggtc accaggttac cgttctgggt 120 gaaatcaaaa ccggtaactctccggttaaa cagtacttct acgaaacccg ttgcaaagaa 180 gctgcaccgg ttgacaacggttgccgtggt atcgacgaca aacactggaa ctctcagtgc 240 aaaacctctc agacctacgttcgtgctctg acctctgaaa acaacaagct tgttggttgg 300 cgttggattc gtatcgacacctcttgcgtt tgcgctctgt ctcgtaaaat cggt 354 5 118 PRT Artificial SequenceDescription of Artificial Sequence Analog of human NT-3. 5 Met Tyr AlaGlu His Lys Ser His Arg Gly Glu Tyr Ser Val Cys Asp 1 5 10 15 Ser GluSer Leu Trp Val Thr Asp Lys Ser Ser Ala Ile Asp Ile Arg 20 25 30 Gly HisGln Val Thr Val Leu Gly Glu Ile Lys Thr Gly Asn Ser Pro 35 40 45 Val LysGln Tyr Phe Tyr Glu Thr Arg Cys Lys Glu Ala Ala Pro Val 50 55 60 Asp AsnGly Cys Arg Gly Ile Asp Asp Lys His Trp Asn Ser Gln Cys 65 70 75 80 LysThr Ser Gln Thr Tyr Val Arg Ala Leu Thr Ser Glu Asn Asn Lys 85 90 95 LeuVal Gly Trp Arg Trp Ile Arg Ile Asp Thr Ser Cys Val Cys Ala 100 105 110Leu Ser Arg Lys Ile Gly 115 6 119 PRT Artificial Sequence Description ofArtificial Sequence Analog of human NT-3. 6 Tyr Ala Glu His Lys Ser HisArg Gly Glu Tyr Ser Val Cys Asp Ser 1 5 10 15 Glu Ser Leu Trp Val ThrAsp Lys Ser Ser Ala Ile Asp Ile Arg Gly 20 25 30 His Gln Val Thr Val LeuGly Glu Ile Lys Thr Gly Asn Ser Pro Val 35 40 45 Lys Gln Tyr Phe Tyr GluThr Arg Cys Lys Glu Ala Ala Pro Val Asp 50 55 60 Asn Gly Cys Arg Gly IleAsp Asp Lys His Trp Asn Ser Gln Cys Lys 65 70 75 80 Thr Ser Gln Thr TyrVal Arg Ala Leu Thr Ser Glu Asn Asn Lys Leu 85 90 95 Val Gly Trp Arg TrpIle Arg Ile Asp Thr Ser Cys Val Cys Ala Leu 100 105 110 Ser Arg Lys IleGly Arg Thr 115 7 117 PRT Artificial Sequence Description of ArtificialSequence Analog of human NT-3. 7 Tyr Ala Glu His Lys Ser His Arg Gly GluTyr Ser Val Cys Asp Ser 1 5 10 15 Glu Ser Leu Trp Val Thr Asp Lys SerSer Ala Ile Asp Ile Arg Gly 20 25 30 His Gln Val Thr Val Leu Gly Glu IleLys Thr Gly Asn Ser Pro Val 35 40 45 Lys Gln Tyr Phe Tyr Glu Thr Arg CysLys Glu Ala Ala Pro Val Asp 50 55 60 Asn Gly Cys Arg Gly Ile Asp Asp LysHis Trp Asn Ser Gln Cys Lys 65 70 75 80 Thr Ser Gln Thr Tyr Val Arg AlaLeu Thr Ser Glu Asn Asn Lys Leu 85 90 95 Val Gly Trp Arg Trp Ile Arg IleAsp Thr Ser Cys Val Cys Ala Leu 100 105 110 Ser Arg Lys Ile Gly 115 8663 DNA Artificial Sequence Description of Artificial Sequence Hybrid ofbacterial (E. coli) and human (Homo sapiens) sequence. 8 cgtaacgtatgcatggtctc cccatgcgag agtagggaac tgccaggcat caataaaacg 60 aaaggctcagtcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgacgctctc 120 ctgagtaggacaaatccgcc gggagcggat ttgaacgttg cgaagcaacg gccggagggt 180 ggcgggcaggacgcccgcca taaactgcca ggcatcaaat taagcagaag ccatcctgac 240 ggatggcctttttgcgtttc tacaaactct tttgtttatt tttctaaata cattcaaata 300 tggacgtctcataattttta aaaaattcat ttgacaaatg ctaaaattct tgattaatat 360 tctcaattgtgagcgctcac aatttatcga tttgattcta gatttgagtt ttaactttta 420 gaaggaggaataacatatgg ttaacgcgtt ggaattcgag ctcactagtg tcgacctgca 480 gggtaccatggaagcttact cgaggatccg cggaaagaag aagaagaaga agaaagcccg 540 aaaggaagctgagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc 600 ctctaaacgggtcttgaggg gttttttgct gaaaggagga accgctcttc acgctcttca 660 cgc 663 9 665DNA Artificial Sequence Description of Artificial Sequence Hybrid ofbacterial (E. coli) and human (Homo sapiens) sequence. 9 gtgaagagcgtgaagagcgg ttcctccttt cagcaaaaaa cccctcaaga cccgtttaga 60 ggccccaaggggttatgcta gttattgctc agcggtggca gcagccaact cagcttcctt 120 tcgggctttcttcttcttct tcttctttcc gcggatcctc gagtaagctt ccatggtacc 180 ctgcaggtcgacactagtga gctcgaattc caacgcgtta accatatgtt attcctcctt 240 ctaaaagttaaaactcaaat ctagaatcaa atcgataaat tgtgagcgct cacaattgag 300 aatattaatcaagaatttta gcatttgtca aatgaatttt ttaaaaatta tgagacgtcc 360 atatttgaatgtattagaaa aataaacaaa agagtttgta gaaacgcaaa aaggccatcc 420 gtcaggatggccttctgctt aatttgatgc ctggcagttt atggcgggcg tcctgcccgc 480 caccctccggccgttgcttc gcaacgttca aatccgctcc cggcggattt gtcctactca 540 ggagagcgtcaccgacaaac aacagataaa acgaaaggcc cagtctttcg actgagcctt 600 tcgttttattgatgcctggc agttccctac tctcgcatgg ggagaccatg catacgttac 660 gcacg 665

What is claimed is:
 1. An isolated polypeptide analog of neurotrophin-3(NT-3) wherein the analog has an amino acid sequence selected from thegroup consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ IDNO:7, and wherein the isoelectric point is lower and the in vivocirculating life and/or absorption is increased for said analog relativeto those same properties in NT-3 of the native amino acid sequence. 2.An isolated polypeptide according to claim 1, which is alsocharacterized by a lower charge under physiogical conditions compared toNT-3 of the native sequence.
 3. An isolated polypeptide according toclaim 1, having the amino acid sequence of SEQ ID NO:
 3. 4. An isolatedpolypeptide according to claim 1, having the amino acid sequence of SEQID NO:
 5. 5. An isolated polypeptide according to claim 1, having theamino acid sequence of SEQ ID NO:
 6. 6. An isolated polypeptideaccording to claim 1, having the amino acid sequence of SEQ ID NO:
 7. 7.A pegylated derivative of a polypeptide according to claim
 1. 8. Apharmaceutical composition comprising a therapeutically effective amountof a polypeptide according to claim 1 and a pharmaceutically acceptablecarrier or diluent.